Primate Central Nervous System Research Articles

Targeting the transferrin receptor to transport antisense oligonucleotides across the mammalian blood-brain barrier.

Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.

Expansion modes of primate nervous system structures in the light of the Prosomeric Model

The expansion of human and non-human primate central nervous system structures has been a paramount question for classic and contemporary studies in comparative vertebrate neuroanatomy. These studies can benefit from framing data analysis within the Prosomeric Model, which defines a common Bauplan for all vertebrate species, including mammals. According to this model, the vertebrate nervous system is composed of several Fundamental Morphological Units (FMUs) that are defined and delineated by characteristic gene expression profiles. Thus, the expansion of neural structures can be traced back to heterochronic neurogenesis, cell lineage specification, and axon growth in their corresponding FMUs. In the present article, we exemplify the use of the Prosomeric Model as the proper theoretical framework for analyzing the expansion of the cerebral and cerebellar cortices, the pontine nuclei, the striatum, the nigrostriatal dopaminergic system, the thalamus, and the amygdala in primates compared to rodents. We describe the quantitative (volume and neuron number) and qualitative (cytoarchitectonic and cell type differences) expansion of these structures in primates versus rodents and define different expansion modes. Then, we relate these modes to the developmental primary events of specification and secondary events of histogenesis, like neurogenesis. We conclude that the systematic analysis of the molecular regulation of primary and secondary developmental events in each FMU in rats, primates, and other mammals could provide the necessary insight to identify the causal mechanisms of the expansion modes described in the present article.

Cellular Localization and Distribution of TGF-β1, GDNF and PDGF-BB in the Adult Primate Central Nervous System.

The available data on the localization of transforming growth factor beta1 (TGF-β1), glial cell line-derived neurotrophic factor (GDNF), and platelet-derived growth factor-BB (PDGF-BB) in the adult primate and human central nervous system (CNS) are limited and lack comprehensive and systematic information. This study aimed to investigate the cellular localization and distribution of TGF-β1, GDNF, and PDGF-BB in the CNS of adult rhesus macaque (Macaca mulatta). Seven adult rhesus macaques were included in the study. The protein levels of TGF-β1, PDGF-BB, and GDNF in the cerebral cortex, cerebellum, hippocampus, and spinal cord were analyzed by western blotting. The expression and location of TGF-β1, PDGF-BB, and GDNF in the brain and spinal cord was examined by immunohistochemistry and immunofluorescence staining, respectively. The mRNA expression of TGF-β1, PDGF-BB, and GDNF was detected by in situ hybridization. The molecular weight of TGF-β1, PDGF-BB, and GDNF in the homogenate of spinal cord was 25 KDa, 30 KDa, and 34 KDa, respectively. Immunolabeling revealed GDNF was ubiquitously distributed in the cerebral cortex, hippocampal formation, basal nuclei, thalamus, hypothalamus, brainstem, cerebellum, and spinal cord. TGF-β1 was least distributed and found only in the medulla oblongata and spinal cord, and PDGF-BB expression was also limited and present only in the brainstem and spinal cord. Besides, TGF-β1, PDGF-BB, and GDNF were localized in the astrocytes and microglia of spinal cord and hippocampus, and their expression was mainly found in the cytoplasm and primary dendrites. The mRNA of TGF-β1, PDGF-BB, and GDNF was localized to neuronal subpopulations in the spinal cord and cerebellum. These findings suggest that TGF-β1, GDNF and PDGF-BB may be associated with neuronal survival, neural regeneration and functional recovery in the CNS of adult rhesus macaques, providing the potential insights into the development or refinement of therapies based on these factors.

AAVrh10 vector corrects pathology in animal models of GM1 gangliosidosis and achieves widespread distribution in the CNS of nonhuman primates

GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes the lysosomal hydrolase acid β-galactosidase (β-gal). β-gal deficiency leads to toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS), resulting in progressive neurodegeneration. LYS-GM101 is an AAVrh.10-based gene therapy vector carrying the human GLB1 cDNA. The efficacy of intra-cerebrospinal fluid injection of LYS-GM101 analogs was demonstrated in GM1 mouse and cat models with widespread diffusion of β-gal and correction of GM1 ganglioside accumulation in the CNS without observable adverse effects. Clinical dose selection was performed, based on a good-laboratory-practice study, in nonhuman primates (NHPs) using the clinical LYS-GM101 vector. A broadly distributed increase of β-gal activity was observed in NHP brain 3 months after intra-cisterna magna injection of LYS-GM101 at 1.0×1012 vg/mL CSF and 4.0×1012 vg/mL CSF, with 20% and 60% increases compared with vehicle-treated animals, respectively. Histopathologic examination revealed asymptomatic adverse changes in the sensory pathways of the spinal cord and dorsal root ganglia in both sexes and at both doses. Taken as a whole, these pre-clinical data support the initiation of a clinical study with LYS-GM101 for the treatment of GM1 gangliosidosis.

Safe and Effective Cynomolgus Monkey GLP-Tox Study with Repetitive Intrathecal Application of a TGFBR2 Targeting LNA-Gapmer Antisense Oligonucleotide as Treatment Candidate for Neurodegenerative Disorders.

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFβ system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide “NVP-13”, targeting TGFBR2, effectively reduced its expression and lowered TGFβ signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13.

Small phenolic and indolic gut-dependent molecules in the primate central nervous system: levels vs. bioactivity.

A rapidly growing body of data documents associations between disease of the brain and small molecules generated by gut-microbiota (GMB). While such metabolites can affect brain function through a variety of mechanisms, the most direct action would be on the central nervous system (CNS) itself. Identify indolic and phenolic GMB-dependent small molecules that reach bioactive concentrations in primate CNS. We conducted a PubMed search for metabolomic studies of the primate CNS [brain tissue or cerebrospinal fluid (CSF)] and then selected for phenolic or indolic metabolites that (i) had been quantified, (ii) were GMB-dependent. For each chemical we then conducted a search for studies of bioactivity conducted in vitro in human cells of any kind or in CNS cells from the mouse or rat. 36 metabolites of interests were identified in primate CNS through targeted metabolomics. Quantification was available for 31/36 and in vitro bioactivity for 23/36. The reported CNS range for 8 metabolites 2-(3-hydroxyphenyl)acetic acid, 2-(4-hydroxyphenyl)acetic acid, 3-(3-hydroxyphenyl)propanoic acid, (E)-3-(3,4-dihydroxyphenyl)prop-2-enoic acid [caffeic acid], 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2-acetamido-3-(1H-indol-3-yl)propanoic acid [N-acetyltryptophan], 1H-indol-3-yl hydrogen sulfate [indoxyl-3-sulfate] overlapped with a bioactive concentration. However, the number and quality of relevant studies of CNS neurochemistry as well as of bioactivity were highly limited. Structural isomers, multiple metabolites and potential confounders were inadequately considered. The potential direct bioactivity of GMB-derived indolic and phenolic molecules on primate CNS remains largely unknown. The field requires additional strategies to identify and prioritize screening of the most promising small molecules that enter the CNS.

The neuronal retromer can regulate both neuronal and microglial phenotypes of Alzheimer's disease.

SUMMARYDisruption of retromer-dependent endosomal trafficking is considered pathogenic in late-onset Alzheimer’s disease (AD). Here, to investigate this disruption in the intact brain, we turn to a genetic mouse model where the retromer core protein VPS35 is depleted in hippocampal neurons, and then we replete VPS35 using an optimized viral vector protocol. The VPS35 depletion-repletion studies strengthen the causal link between the neuronal retromer and AD-associated neuronal phenotypes, including the acceleration of amyloid precursor protein cleavage and the loss of synaptic glutamate receptors. Moreover, the studies show that the neuronal retromer can regulate a distinct, dystrophic, microglia morphology, phenotypic of hippocampal microglia in AD. Finally, the neuronal and, in part, the microglia responses to VPS35 depletion were found to occur independent of tau. Showing that the neuronal retromer can regulate AD-associated pathologies in two of AD’s principal cell types strengthens the link, and clarifies the mechanism, between endosomal trafficking and late-onset sporadic AD.

Light microscopic and heterogeneity analysis of astrocytes in the common marmoset brain.

Astrocytes are abundant cells of the central nervous system (CNS) and are involved in processes including synapse formation/function, ion homeostasis, neurotransmitter uptake, and neurovascular coupling. Recent evidence indicates that astrocytes show diverse molecular, structural, and physiological properties within the CNS. This heterogeneity is reflected in differences in astrocyte structure, gene expression, functional properties, and responsiveness to injury/pathological conditions. Deeper investigation of astrocytic heterogeneity is needed to understand how astrocytes are configured to enable diverse roles in the CNS. While much has been learned about astrocytic heterogeneity in rodents, much less is known about astrocytic heterogeneity in the primate brain where astrocytes have greater size and complexity. The common marmoset (Callithrix jacchus) is a promising non‐human primate model because of similarities between marmosets and humans with respect to genetics, brain anatomy, and cognition/behavior. Here, we investigated the molecular and structural heterogeneity of marmoset astrocytes using an array of astrocytic markers, multi‐label confocal microscopy, and quantitative analysis. We used male and female marmosets and found that marmoset astrocytes show differences in expression of astrocytic markers in cortex, hippocampus, and cerebellum. These differences were accompanied by intra‐regional variation in expression of markers for glutamate/GABA transporters, and potassium and water channels. Differences in astrocyte structure were also found, along with complex interactions with blood vessels, microglia, and neurons. This study contributes to our knowledge of the cellular and molecular features of marmoset astrocytes and is useful for understanding the complex properties of astrocytes in the primate CNS.

Nonhuman primates at the intersection of aging biology, chronic disease, and health: An introduction to the American Journal of Primatology Special Issue on aging, cognitive decline, and neuropathology in nonhuman primates.

Aging across the Primate Order is poorly understood because ages of individuals are often unknown, there is a dearth of aged animals available for study, and because aging is best characterized by longitudinal studies which are difficult to carry out in long-lived species. The human population is aging rapidly, and advanced age is a primary risk factor for several chronic diseases and conditions that impact healthspan. As lifespan has increased, diseases and disorders of the central nervous system (CNS) have become more prevalent, and Alzheimer's disease and related dementias have become epidemic. Nonhuman primate (NHP) models are key to understanding the aging primate CNS. This Special Issue presents a review of current knowledge about NHP CNS aging across the Primate Order. Similarities and differences to human aging, and their implications for the validity of NHP models of aging are considered. Topics include aging-related brain structure and function, neuropathologies, cognitive performance, social behavior and social network characteristics, and physical, sensory, and motor function. Challenges to primate CNS aging research are discussed. Together, this collection of articles demonstrates the value of studying aging in a breadth of NHP models to advance our understanding of human and nonhuman primate aging and healthspan.

A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system

Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain. Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain.

Bundle-Specific Axon Diameter Index as a New Contrast to Differentiate White Matter Tracts.

In the central nervous system of primates, several pathways are characterized by different spectra of axon diameters. In vivo methods, based on diffusion-weighted magnetic resonance imaging, can provide axon diameter index estimates non-invasively. However, such methods report voxel-wise estimates, which vary from voxel-to-voxel for the same white matter bundle due to partial volume contributions from other pathways having different microstructure properties. Here, we propose a novel microstructure-informed tractography approach, COMMITAxSize, to resolve axon diameter index estimates at the streamline level, thus making the estimates invariant along trajectories. Compared to previously proposed voxel-wise methods, our formulation allows the estimation of a distinct axon diameter index value for each streamline, directly, furnishing a complementary measure to the existing calculation of the mean value along the bundle. We demonstrate the favourable performance of our approach comparing our estimates with existing histologically-derived measurements performed in the corpus callosum and the posterior limb of the internal capsule. Overall, our method provides a more robust estimation of the axon diameter index of pathways by jointly estimating the microstructure properties of the tissue and the macroscopic organisation of the white matter connectivity.

Safety of Direct Intraparenchymal AAVrh.10-Mediated Central Nervous System Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy, a fatal pediatric neurodegenerative lysosomal storage disease caused by mutations in the arylsulfatase A (ARSA) gene, is characterized by intracellular accumulation of sulfatides in the lysosomes of cells of the central nervous system (CNS). In previous studies, we have demonstrated efficacy of AAVrh.10hARSA, an adeno-associated virus (AAV) serotype rh.10 vector coding for the human ARSA gene to the CNS of a mouse model of the disease, and that catheter-based intraparenchymal administration of AAVrh.10hARSA to the CNS of nonhuman primates (NHPs) white matter results in widespread expression of ARSA. As a formal dose-escalating safety/toxicology study, we assessed the safety of intraparenchymal delivery of AAVrh.10hARSA vector to 12 sites in the white matter of the CNS of NHPs at 2.85 × 1010 (total low dose, 2.4 × 109 genome copies [gc]/site) and 1.5 × 1012 (total high dose, 1.3 × 1011 gc/site) gc, compared to AAVrh.10Null (1.5 × 1012 gc total, 1.3 × 1011 gc/site) as a vector control, and phosphate buffered saline for a sham surgical control. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA. However, animals treated with the high dose AAVrh.10ARSA and the high dose Null vector had highly localized CNS abnormalities on magnetic resonance imaging scans at the sites of catheter infusions, and histopathology demonstrated that these sites were associated with infiltrates of T cells, B cells, microglial cells, and/or macrophages. Although these findings had no clinical consequences, these safety data contribute to understanding the dose limits for CNS white matter direct intraparenchymal administration of AAVrh.10 vectors for treatment of CNS disorders.

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

Central Nervous System Migration of Astaxanthin and Adonixanthin Following Their Oral Administration in Cynomolgus Monkeys.

Astaxanthin, which has been shown to have significant antioxidant activity, is rapidly spreading as a health functioning ingredient in the health food and cosmetics sectors worldwide. It is well known that astaxanthin acts on the brain; however, there is little evidence of brain translocation due to the difficulty in identifying astaxanthin in tissues. Therefore, in this study, we investigated the concentrations of astaxanthin and adonixanthin, the latter being a biosynthetic intermediate from β-carotene to astaxanthin, in the brain after oral administration in primates. Cynomolgus monkeys were orally administered astaxanthin or adonixanthin at a dose of 50 mg/kg for 10 d, through a disposable catheter inserted into the stomach via the nasal passage. Following euthanization, the monkeys' brains and various other organs were collected. The carotenoid content in serum and individual organs was analyzed by high-performance liquid chromatography. Adonixanthin was found to accumulate at a higher concentration than astaxanthin in monkey brain tissues. Also, both astaxanthin and adonixanthin were found to be distributed in the heart, spleen, liver, and kidneys. These findings indicate that astaxanthin and adonixanthin can enter the central nervous system of primates following their oral administration. This provides important evidence for the activity of astaxanthin and adonixanthin on the central nervous system.

Species-Specific miRNAs in Human Brain Development and Disease.

Identification of the unique features of human brain development and function can be critical towards the elucidation of intricate processes such as higher cognitive functions and human-specific pathologies like neuropsychiatric and behavioral disorders. The developing primate and human central nervous system (CNS) are distinguished by expanded progenitor zones and a protracted time course of neurogenesis, leading to the expansion in brain size, prominent gyral anatomy, distinctive synaptic properties, and complex neural circuits. Comparative genomic studies have revealed that adaptations of brain capacities may be partly explained by human-specific genetic changes that impact the function of proteins associated with neocortical expansion, synaptic function, and language development. However, the formation of complex gene networks may be most relevant for brain evolution. Indeed, recent studies identified distinct human-specific gene expression patterns across developmental time occurring in brain regions linked to cognition. Interestingly, such modules show species-specific divergence and are enriched in genes associated with neuronal development and synapse formation whilst also being implicated in neuropsychiatric diseases. microRNAs represent a powerful component of gene-regulatory networks by promoting spatiotemporal post-transcriptional control of gene expression in the human and primate brain. It has also been suggested that the divergence in miRNA expression plays an important role in shaping gene expression divergence among species. Primate-specific and human-specific miRNAs are principally involved in progenitor proliferation and neurogenic processes but also associate with human cognition, and neurological disorders. Human embryonic or induced pluripotent stem cells and brain organoids, permitting experimental access to neural cells and differentiation stages that are otherwise difficult or impossible to reach in humans, are an essential means for studying species-specific brain miRNAs. Single-cell sequencing approaches can further decode refined miRNA-mRNA interactions during developmental transitions. Elucidating species-specific miRNA regulation will shed new light into the mechanisms that control spatiotemporal events during human brain development and disease, an important step towards fostering novel, holistic and effective therapeutic approaches for neural disorders. In this review, we discuss species-specific regulation of miRNA function, its contribution to the evolving features of the human brain and in neurological disease, with respect also to future therapeutic approaches.

Transduction of Craniofacial Motoneurons Following Intramuscular Injections of Canine Adenovirus Type-2 (CAV-2) in Rhesus Macaques.

Reliable viral vector-mediated transgene expression in primate motoneurons would improve our ability to anatomically and physiologically interrogate motor systems. We therefore investigated the efficacy of replication defective, early region 1-deleted canine adenovirus type-2 (CAV-2) vectors for mediating transgene expression of fluorescent proteins into brainstem motoneurons following craniofacial intramuscular injections in four rhesus monkeys (Macaca mulatta). Vector injections were placed into surgically identified and isolated craniofacial muscles. After a 1- to 2-month survival time, animals were sacrificed and transgene expression was assessed with immunohistochemistry in the corresponding motoneuronal populations. We found that injections of CAV-2 into individual craniofacial muscles at doses in the range of ∼1010 to 1011 physical particles/muscle resulted in robust motoneuronal transduction and expression of immunohistochemically identified fluorescent proteins across multiple animals. By using different titers in separate muscles, with the resulting transduction patterns tracked via fluorophore expression and labeled motoneuron location, we established qualitative dose-response relationships in two animals. In one animal that received an atypically high titer (5.7 × 1011 total CAV-2 physical particles) distributed across numerous injection sites, no transduction was detected, likely due to a retaliatory immune response. We conclude that CAV-2 vectors show promise for genetic modification of primate motoneurons following craniofacial intramuscular injections. Our findings warrant focused attention toward the use of CAV-2 vectors to deliver opsins, DREADDs, and other molecular probes to improve genetics-based methods for primate research. Further work is required to optimize CAV-2 transduction parameters. CAV-2 vectors encoding proteins could provide a new, reliable route for modifying activity in targeted neuronal populations of the primate central nervous system.

Convection-enhanced delivery of botulinum toxin serotype A into the nonhuman primate cisterna magna and hippocampus.

Botulinum toxin serotype A (BoNT/A) was reported to raise the seizure threshold when injected into the seizure focus of a kindled rodent model. Delivering BoNT/A to the nonhuman primate (NHP) central nervous system via convection-enhanced delivery (CED) has not been performed. The objective of this study was to determine the toxicity and distribution characteristics of CED of BoNT/A into the NHP hippocampus and cisterna magna. Escalating BoNT/A doses were delivered by CED into the NHP hippocampus (n = 4) and cisterna magna (n = 5) for behavioral and histological assessment and to determine the highest nonlethal dose (LD0) and median lethal dose (LD50). Hippocampal BoNT/A was coinfused with Gd-albumin, a surrogate MRI tracer. Gd-albumin and radioiodinated BoNT/A (125I-BoNT/A) were coinfused into the hippocampus of 3 additional NHPs to determine BoNT/A distribution by in vivo MRI and postmortem quantitative autoradiography. Scintillation counting of CSF assessed the flow of 125I-BoNT/A from the hippocampus to CSF postinfusion. LD0 and LD50 were 4.2 and 18 ng/kg, and 5 and > 5 ng/kg for the NHP hippocampus and cisterna magna, respectively. Gd-albumin and 125I-BoNT/A completely perfused the hippocampus (155-234 mm3) in 4 of 7 NHPs. Fifteen percent of BoNT/A entered CSF after hippocampal infusion. The MRI distribution volume of coinfused Gd-albumin (VdMRI) was similar to the quantitative autoradiography distribution of 125I-BoNT/A (VdQAR) (mean VdMRI = 139.5 mm3 [n = 7]; VdQAR = 134.8 mm3 [n = 3]; r = 1.00, p < 0.0001). No infusion-related toxicity was identified histologically except that directly attributable to needle placement. Gd-albumin accurately tracked BoNT/A distribution on MRI. BoNT/A did not produce CNS toxicity. BoNT/A LD0 exceeded 10-fold the dose administered safely to humans for cosmesis and dystonia.

Efficient Delivery of Nerve Growth Factors to the Central Nervous System for Neural Regeneration.

The central nervous system (CNS) plays a central role in the control of sensory and motor functions, and the disruption of its barriers can result in severe and debilitating neurological disorders. Neurotrophins are promising therapeutic agents for neural regeneration in the damaged CNS. However, their penetration across the blood-brain barrier remains a formidable challenge, representing a bottleneck for brain and spinal cord therapy. Herein, a nanocapsule-based delivery system is reported that enables intravenously injected nerve growth factor (NGF) to enter the CNS in healthy mice and nonhuman primates. Under pathological conditions, the delivery of NGF enables neural regeneration, tissue remodeling, and functional recovery in mice with spinal cord injury. This technology can be utilized to deliver other neurotrophins and growth factors to the CNS, opening a new avenue for tissue engineering and the treatment of CNS disorders and neurodegenerative diseases.

Effects of sex and prenatal androgen manipulations on Onuf's nucleus of rhesus macaques

The role of gonadal steroids in sexual differentiation of the central nervous system (CNS) is well established in rodents, but no study to date has manipulated androgens prenatally and examined their effects on any CNS structure in a primate. Onuf's nucleus is a column of motoneurons in the sacral spinal cord that innervates the striated perineal muscles. This cell group is larger in males than in females of many species, due to androgens acting during a sensitive perinatal period. Here, we examined Onuf's nucleus in 21 adult rhesus monkeys, including control males and females, as well as males whose mothers had been treated with an anti-androgen or testosterone during gestation. We found a robust sex difference, with more motoneurons in control males than in females. The soma size of Onuf's nucleus motoneurons was also marginally larger in males. Treatment with the anti-androgen flutamide for 35–40 days during early gestation partially blocked masculinization of Onuf's nucleus: motoneuron number in flutamide-treated males was decreased relative to control and testosterone-treated males, but remained greater than in females, with no effect on cell size. A control motor nucleus that innervates foot muscles (Pes9) showed no difference in motoneuron number or size between control males and females. Prenatal testosterone treatment of males did not alter Onuf's nucleus motoneuron number, but did increase the size of both Onuf's and Pes9 motoneurons. Thus, prenatal androgen manipulations cause cellular-level changes in the primate CNS, which may underlie previously observed effects of these manipulations on behavior.

AAVrh.10-Mediated APOE2 Central Nervous System Gene Therapy for APOE4-Associated Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive degenerative neurological disorder affecting nearly one in nine elderly people in the United States. Population studies have shown that an inheritance of the apolipoprotein E (APOE) variant APOE4 allele increases the risk of developing AD, whereas APOE2 homozygotes are protected from late-onset AD. It was hypothesized that expression of the "protective" APOE2 variant by genetic modification of the central nervous system (CNS) of APOE4 homozygotes could reverse or prevent progressive neurologic damage. To assess the CNS distribution and safety of APOE2 gene therapy for AD in a large-animal model, intraparenchymal, intracisternal, and intraventricular routes of delivery to the CNS of nonhuman primates of AAVrh.10hAPOE2-HA, an AAVrh.10 serotype coding for an HA-tagged human APOE2 cDNA sequence, were evaluated. To evaluate the route of delivery that achieves the widest extent of APOE2 expression in the CNS, the expression of APOE2 in the CNS was evaluated 2 months following vector administration for APOE2 DNA, mRNA, and protein. Finally, using conventional toxicology assays, the safety of the best route of delivery was assessed. The data demonstrated that while all three routes are capable of mediating ApoE2 expression in AD relevant regions, intracisternal delivery of AAVrh.10hAPOE2-HA safely mediated wide distribution of ApoE2 with the least invasive surgical intervention, thus providing the optimal strategy to deliver vector-mediated human APOE2 to the CNS.